Corneal blindness affects about 6-8 million people worldwide. One of its clinically important causes is limbal epithelial stem cell (LESC) deficiency (LSCD). It may be due to genetic diseases, burns, infections, and chronic inflammation and results in corneal scarring, vascularization and conjunctivalization leading to vision loss. Normal LESC transplantation can improve vision. Keratolimbal auto- or allografts approved in U.S. for LSCD have only 30-45% 3-5-year graft survival. In other countries, cultured LESC are transplanted, with a 1-3 years success rate of 76%. However, this procedure also has drawbacks including lack of standardization and low allograft survival, limited number of LESC passages in culture, and routine use of mouse 3T3 feeder layer. To date, there is a clear need for a renewable and standardized source of LESC for treating LSCD. In this proposal, we take a novel approach to this significant clinical problem using induced pluripotent stem cells (iPSC). Unlike LESC, iPSC are immortal, allowing continual propagation and banking. Standard iPSC derivation and growth protocols are amenable to GLP and GMP. We aim at obtaining a renewable LESC source for treating LSCD by generating iPSC and differentiating them back to LESC. We propose for the first time to use LESC cultures to obtain iPSC for redifferentiation back to limbal cells. Our strategy is based on the hypothesis that iPSC differentiation back to LESC may be facilitated by the mechanism of retention by iPSC of parent cell epigenetic signatures. We also propose to use a natural niche for iPSC differentiation to LESC, such as denuded human organ-cultured corneas or amniotic membrane (HAM). HAM is used as matrix for transplantable LESC, but it is tedious to work with and to remove amniotic cells before LESC culture. We developed a novel method for denuding HAM, supporting a reliable growth of LESC and iPSC. These novel approaches backed by our preliminary data may quickly advance the field of LESC transplantation. We hypothesize that making iPSC from limbal cells will facilitate their redifferentiation to LESC via epigenetic memory mechanism. This redifferentiation may be enhanced by LESC extracellular niche such as limbal basement membrane or HAM, combined with growth factors. We aim at obtaining reliable and renewable transplant-grade LESC source. Specific Aim 1. To generate and characterize iPSC from cultured human donor limbal stem cells. Specific Aim 2. To directionally differentiate limbal-derived iPSC back into LESC using a combination of soluble factors and extracellular niche (HAM and denuded human corneas or limbal rims). Specific Aim 3. To achieve transplantation of iPSC-derived limbal cells to the denuded limbal zone of organ-cultured human corneas, to restore normal stem cell compartment structure and function. Health relevance: our novel strategy of obtaining LESC-derived iPSC and differentiating them back to LESC using natural extracellular niche will yield a reliable and renewable source of transplantable LESC for LSCD.